RESUMO
This study isolated a novel peptide MMGGED with strong calcium-binding capacity from defatted walnut meal and synthesized a novel peptidecalcium chelate COS-MMGGED-Ca with high stability via glycation. Structural characterization and computer simulation identified binding sites, while in vitro digestion stability and calcium transport experiments explored the chelate's properties. Results showed that after glycation, COS-MMGGED bound Ca2+ with 88.75 ± 1.75 %, mainly via aspartic and glutamic acids. COS-MMGGED-Ca released Ca2+ steadily (60.27 %), with thermal denaturation temperature increased by 18 °C and 37 °C compared to MMGGED-Ca, indicating good processing performance. Furthermore, COS-MMGGED significantly enhanced Ca2+ transport across Caco-2 monolayers, 1.13-fold and 1.62-fold higher than CaCl2 and MMGGED, respectively, at 240 h. These findings prove glycation enhances structural properties, stability, calcium loading, and transport of peptidecalcium chelates, providing a scientific basis for developing novel efficient calcium supplements and high-value utilization of walnut meal.
Assuntos
Cálcio , Juglans , Peptídeos , Juglans/química , Humanos , Cálcio/química , Cálcio/metabolismo , Células CACO-2 , Peptídeos/química , Peptídeos/metabolismo , Glicosilação , Quelantes de Cálcio/químicaRESUMO
The residual bone tumor and defects which is caused by surgical therapy of bone tumor is a major and important problem in clinicals. And the sequential treatment for irradiating residual tumor and repairing bone defects has wildly prospects. In this study, we developed a general modification strategy by gallic acid (GA)-assisted coordination chemistry to prepare black calcium-based materials, which combines the sequential photothermal therapy of bone tumor and bone defects. The GA modification endows the materials remarkable photothermal properties. Under the near-infrared (NIR) irradiation with different power densities, the black GA-modified bone matrix (GBM) did not merely display an excellent performance in eliminating bone tumor with high temperature, but showed a facile effect of the mild-heat stimulation to accelerate bone regeneration. GBM can efficiently regulate the microenvironments of bone regeneration in a spatial-temporal manner, including inflammation/immune response, vascularization and osteogenic differentiation. Meanwhile, the integrin/PI3K/Akt signaling pathway of bone marrow mesenchymal stem cells (BMSCs) was revealed to be involved in the effect of osteogenesis induced by the mild-heat stimulation. The outcome of this study not only provides a serial of new multifunctional biomaterials, but also demonstrates a general strategy for designing novel blacked calcium-based biomaterials with great potential for clinical use.
Assuntos
Neoplasias Ósseas , Regeneração Óssea , Cálcio , Ácido Gálico , Células-Tronco Mesenquimais , Ácido Gálico/química , Regeneração Óssea/efeitos dos fármacos , Animais , Cálcio/metabolismo , Neoplasias Ósseas/terapia , Neoplasias Ósseas/tratamento farmacológico , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Terapia Fototérmica/métodos , Osteogênese/efeitos dos fármacos , Camundongos , Humanos , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Linhagem Celular TumoralRESUMO
Phytoplankton can encounter dynamic changes in their environment including fluctuating nutrient supply, and therefore require survival mechanisms to compete for such growth-limiting resources. Diatoms, single-celled eukaryotic microalgae, are typically first responders when crucial macronutrients phosphorus (P) and nitrogen (N) enter the marine environment and therefore must have tightly regulated nutrient sensory systems. While nutrient starvation responses have been described, comparatively little is known about diatom nutrient sensing mechanisms. We previously identified that the model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana use calcium (Ca2+) ions as a rapid intracellular signaling response following phosphate resupply. This response is evident only in phosphate deplete conditions, suggesting that it is coordinated in P-starved cells. Rapid increases in N uptake and assimilation pathways observed following phosphate resupply, indicate tight interplay between P and N signaling. To regulate such downstream changes, Ca2+ ions must bind to Ca2+ sensors following phosphate induced Ca2+ signals, yet this molecular machinery is unknown. Here, we describe our findings in relation to known diatom P starvation signaling mechanisms and discuss their implications in the context of environmental macronutrient metadata and in light of recent developments in the field. We also consider the importance of studying phytoplankton nutrient signaling systems in the face of future ocean conditions.
Assuntos
Diatomáceas , Nitrogênio , Fosfatos , Diatomáceas/metabolismo , Nitrogênio/metabolismo , Fosfatos/metabolismo , Cálcio/metabolismo , Transdução de Sinais , Fitoplâncton/metabolismoRESUMO
Many mammal species have declining populations, but the consequences of small population size on the genomic makeup of species remain largely unknown. We investigated the evolutionary history, genetic load and adaptive potential of the Cat Ba langur (Trachypithecus poliocephalus), a primate species endemic to Vietnam's famous Ha Long Bay and with less than 100 living individuals one of the most threatened primates in the world. Using high-coverage whole genome data of four wild individuals, we revealed the Cat Ba langur as sister species to its conspecifics of the northern limestone langur clade and found no evidence for extensive secondary gene flow after their initial separation. Compared to other primates and mammals, the Cat Ba langur showed low levels of genetic diversity, long runs of homozygosity, high levels of inbreeding and an excess of deleterious mutations in homozygous state. On the other hand, genetic diversity has been maintained in protein-coding genes and on the gene-rich human chromosome 19 ortholog, suggesting that the Cat Ba langur retained most of its adaptive potential. The Cat Ba langur also exhibits several unique non-synonymous variants that are related to calcium and sodium metabolism, which may have improved adaptation to high calcium intake and saltwater consumption.
Assuntos
Espécies em Perigo de Extinção , Variação Genética , Densidade Demográfica , Animais , Vietnã , Adaptação Fisiológica/genética , Genoma/genética , Filogenia , Cromossomos Humanos Par 19/genética , Homozigoto , Humanos , Fluxo Gênico , Genômica/métodos , Endogamia , Cálcio/metabolismoRESUMO
Inulin, a widely recognized prebiotic, has diverse applications across various industrial sectors. Although inulin is primarily produced through plant extraction, there is growing interest in enzymatic synthesis as an alternative. The enzymatic production of inulin from sucrose, which yields polymers with degrees of polymerization similar to those of plant-derived inulin, shows potential as a viable replacement for traditional extraction methods. In this study, an inulosucrase from Neobacillus bataviensis was identified, demonstrating a non-processive mechanism specifically tailored for synthesizing inulin with polymerization degrees ranging from 3 to approximately 40. The enzyme exhibited optimal activity at pH 6.5 and 55 °C, efficiently producing inulin with a yield of 50.6%. Ca2+ can improve the activity and thermostability of this enzyme. To enhance catalytic total activity, site-directed and truncated mutagenesis techniques were applied, resulting in the identification of a mutant, T149S, displaying a significant 57% increase in catalytic total activity. Molecular dynamics simulations unveiled that the heightened flexibility observed in three surface regions positively influenced enzymatic activity. This study not only contributes to the theoretical foundation for inulosucrase engineering but also presents a potential avenue for the production of inulin.
Assuntos
Hexosiltransferases , Inulina , Inulina/metabolismo , Hexosiltransferases/metabolismo , Hexosiltransferases/genética , Hexosiltransferases/química , Simulação de Dinâmica Molecular , Concentração de Íons de Hidrogênio , Sacarose/metabolismo , Peso Molecular , Mutagênese Sítio-Dirigida , Bacillales/enzimologia , Bacillales/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Engenharia de Proteínas , Estabilidade Enzimática , Temperatura , Cálcio/metabolismoRESUMO
Frequent interspecies transmission of human influenza A viruses (FLUAV) to pigs contrasts with the limited subset that establishes in swine. While hemagglutinin mutations are recognized for their role in cross-species transmission, the contribution of neuraminidase remains understudied. Here, the NA's role in FLUAV adaptation was investigated using a swine-adapted H3N2 reassortant virus with human-derived HA and NA segments. Adaptation in pigs resulted in mutations in both HA (A138S) and NA (D113A). The D113A mutation abolished calcium (Ca2+) binding in the low-affinity Ca2+-binding pocket of NA, enhancing enzymatic activity and thermostability under Ca2+-depleted conditions, mirroring swine-origin FLUAV NA behavior. Structural analysis predicts that swine-adapted H3N2 viruses lack Ca2+ binding in this pocket. Further, residue 93 in NA (G93 in human, N93 in swine) also influences Ca2+ binding and impacts NA activity and thermostability, even when D113 is present. These findings demonstrate that mutations in influenza A virus surface proteins alter evolutionary trajectories following interspecies transmission and reveal distinct mechanisms modulating NA activity during FLUAV adaptation, highlighting the importance of Ca2+ binding in the low-affinity calcium-binding pocket.
Assuntos
Cálcio , Neuraminidase , Neuraminidase/metabolismo , Neuraminidase/genética , Neuraminidase/química , Humanos , Animais , Cálcio/metabolismo , Suínos , Sítios de Ligação , Infecções por Orthomyxoviridae/virologia , Infecções por Orthomyxoviridae/transmissão , Influenza Humana/virologia , Influenza Humana/transmissão , Adaptação Fisiológica/genética , Proteínas Virais/metabolismo , Proteínas Virais/genética , Proteínas Virais/química , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/metabolismo , Mutação , Ligação Proteica , Doenças dos Suínos/virologiaRESUMO
BACKGROUND: The formation of stem cell clones enables close contact of stem cells inside. The gap junctions in such clone spheres establish a microenvironment that allows frequent intercellular communication to maintain self-renewal and functions of stem cells. Nevertheless, the essential gap junction protein for molecular signaling in clones is poorly known. METHODS: Primary human airway basal cells (hBCs) were isolated from brushing samples through bronchoscopy and then cultured. A tightly focused femtosecond laser was used to excite the local Ca2+ in an individual cell to initiate an internal Ca2+ wave in a clone to screen gap junction proteins. Immunoflourescence staining and clonogenicity assay were used to evaluate self-renewal and functions. RNA and protein levels were assessed by PCR and Western blot. Air-liquid interface assay was conducted to evaluate the differentiation potential. A Naphthalene injury mouse model was used to assess the regeneration potential. RESULTS: Herein, we identify Connexin 25 (Cx25) dominates intercellular Ca2+ communications in clones of hBCs in vitro to maintain the self-renewal and pluripotency of them. The self-renewal and in vitro differentiation functions and in vivo regeneration potential of hBCs in an airway damage model are both regulated by Cx25. The abnormal expression of Cx25 is validated in several diseases including IPF, Covid-19 and bronchiectasis. CONCLUSION: Cx25 is essential for hBC clones in maintaining self-renewal and functions of hBCs via gap junctions.
Assuntos
Conexinas , Regeneração , Humanos , Animais , Camundongos , Conexinas/metabolismo , Conexinas/genética , Diferenciação Celular , COVID-19/metabolismo , COVID-19/virologia , COVID-19/patologia , Junções Comunicantes/metabolismo , Autorrenovação Celular , Cálcio/metabolismo , Células Cultivadas , SARS-CoV-2/metabolismo , Masculino , Células-Tronco/metabolismo , Células-Tronco/citologiaRESUMO
The molecular mechanisms leading to saliva secretion are largely established, but factors that underlie secretory hypofunction, specifically related to the autoimmune disease Sjögren's syndrome (SS) are not fully understood. A major conundrum is the lack of association between the severity of salivary gland immune cell infiltration and glandular hypofunction. SS-like disease was induced by treatment with DMXAA, a small molecule agonist of murine STING. We have previously shown that the extent of salivary secretion is correlated with the magnitude of intracellular Ca2+ signals (Takano et al., 2021). Contrary to our expectations, despite a significant reduction in fluid secretion, neural stimulation resulted in enhanced Ca2+ signals with altered spatiotemporal characteristics in vivo. Muscarinic stimulation resulted in reduced activation of the Ca2+-activated Cl- channel, TMEM16a, although there were no changes in channel abundance or absolute sensitivity to Ca2+. Super-resolution microscopy revealed a disruption in the colocalization of Inositol 1,4,5-trisphosphate receptor Ca2+ release channels with TMEM16a, and channel activation was reduced when intracellular Ca2+ buffering was increased. These data indicate altered local peripheral coupling between the channels. Appropriate Ca2+ signaling is also pivotal for mitochondrial morphology and bioenergetics. Disrupted mitochondrial morphology and reduced oxygen consumption rate were observed in DMXAA-treated animals. In summary, early in SS disease, dysregulated Ca2+ signals lead to decreased fluid secretion and disrupted mitochondrial function contributing to salivary gland hypofunction.
Assuntos
Anoctamina-1 , Sinalização do Cálcio , Modelos Animais de Doenças , Mitocôndrias , Síndrome de Sjogren , Animais , Síndrome de Sjogren/metabolismo , Camundongos , Mitocôndrias/metabolismo , Anoctamina-1/metabolismo , Cálcio/metabolismo , Glândulas Salivares/metabolismo , Glândulas Salivares/patologia , Feminino , Camundongos Endogâmicos C57BLRESUMO
The clinical management of severe COVID-19 cases is not yet well resolved. Therefore, it is important to identify and characterize cell signaling pathways involved in virus pathogenesis that can be targeted therapeutically. Envelope (E) protein is a structural protein of the virus, which is known to be highly expressed in the infected host cell and is a key virulence factor; however, its role is poorly characterized. The E protein is a single-pass transmembrane protein that can assemble into a pentamer forming a viroporin, perturbing Ca2+ homeostasis. Because it is structurally similar to regulins such as, for example, phospholamban, that regulate the sarco/endoplasmic reticulum calcium ATPases (SERCA), we investigated whether the SARS-CoV-2 E protein affects the SERCA system as an exoregulin. Using FRET experiments we demonstrate that E protein can form oligomers with regulins, and thus can alter the monomer/multimer regulin ratio and consequently influence their interactions with SERCAs. We also confirm that a direct interaction between E protein and SERCA2b results in a decrease in SERCA-mediated ER Ca2+ reload. Structural modeling of the complexes indicates an overlapping interaction site for E protein and endogenous regulins. Our results reveal novel links in the host-virus interaction network that play an important role in viral pathogenesis and may provide a new therapeutic target for managing severe inflammatory responses induced by SARS-CoV-2.
Assuntos
COVID-19 , Sinalização do Cálcio , Proteínas do Envelope de Coronavírus , SARS-CoV-2 , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Humanos , SARS-CoV-2/metabolismo , COVID-19/virologia , COVID-19/metabolismo , Proteínas do Envelope de Coronavírus/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Ligação ProteicaRESUMO
The inducers of neutrophil extracellular trap (NET) formation are heterogeneous and consequently, there is no specific pathway or signature molecule indispensable for NET formation. But certain events such as histone modification, chromatin decondensation, nuclear envelope breakdown, and NET release are ubiquitous. During NET formation, neutrophils drastically rearrange their cytoplasmic, granular and nuclear content. Yet, the exact mechanism for decoding each step during NET formation still remains elusive. Here, we investigated the mechanism of nuclear envelope breakdown during NET formation. Immunofluorescence microscopic evaluation revealed a gradual disintegration of outer nuclear membrane protein nesprin-1 and alterations in nuclear morphology during NET formation. MALDI-TOF analysis of NETs that had been generated by various inducers detected the accumulation of nesprin-1 fragments. This suggests that nesprin-1 degradation occurs before NET release. In the presence of a calpain-1, inhibitor nesprin-1 degradation was decreased in calcium driven NET formation. Microscopic evaluation confirmed that the disintegration of the lamin B receptor (LBR) and the collapse of the actin cytoskeleton occurs in early and later phases of NET release, respectively. We conclude that the calpain-1 degrades nesprin-1, orchestrates the weakening of the nuclear membrane, contributes to LBR disintegration, and promoting DNA release and finally, NETs formation.
Assuntos
Calpaína , Armadilhas Extracelulares , Receptor de Lamina B , Neutrófilos , Membrana Nuclear , Membrana Nuclear/metabolismo , Calpaína/metabolismo , Humanos , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Cálcio/metabolismo , Proteínas do CitoesqueletoRESUMO
G-protein-coupled receptor kinase 2 (GRK2) interacts with Gßγ and Gαq, subunits of G-protein alpha, to regulate cell signalling. The second messenger inositol trisphosphate, produced by activated Gαq, promotes calcium release from the endoplasmic reticulum (ER) and regulates maturation-promoting factor (MPF) activity. This study aimed to investigate the role of GRK2 in MPF activity during the meiotic maturation of porcine oocytes. A specific inhibitor of GRK2 (ßi) was used in this study. The present study showed that GRK2 inhibition increased the percentage of oocyte arrest at the metaphase I (MI) stage (control: 13.84 ± 0.95%; ßi: 31.30 ± 4.18%), which resulted in the reduction of the maturation rate (control: 80.36 ± 1.94%; ßi: 65.40 ± 1.14%). The level of phospho-GRK2 decreased in the treated group, suggesting that GRK2 activity was reduced upon GRK2 inhibition. Furthermore, the addition of ßi decreased Ca2+ release from the ER. The protein levels of cyclin B and cyclin-dependent kinase 1 were higher in the treatment group than those in the control group, indicating that GRK2 inhibition prevented a decrease in MPF activity. Collectively, GRK2 inhibition induced meiotic arrest at the MI stage in porcine oocytes by preventing a decrease in MPF activity, suggesting that GRK2 is essential for oocyte meiotic maturation in pigs.
Assuntos
Cálcio , Quinase 2 de Receptor Acoplado a Proteína G , Meiose , Oócitos , Animais , Oócitos/efeitos dos fármacos , Meiose/efeitos dos fármacos , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Feminino , Cálcio/metabolismo , Suínos , Fator Promotor de Maturação/metabolismo , Técnicas de Maturação in Vitro de Oócitos/veterináriaRESUMO
Rationale: Mechanical force plays crucial roles in extracellular vesicle biogenesis, release, composition and activity. However, it is unknown whether mechanical force regulates apoptotic vesicle (apoV) production. Methods: The effects of mechanical unloading on extracellular vesicles of bone marrow were evaluated through morphology, size distribution, yield, and protein mass spectrometry analysis using hindlimb unloading (HU) mouse model. Apoptosis resistance and aging related phenotype were assessed using HU mouse model in vivo and cell microgravity model in vitro. The therapeutic effects of apoVs on HU mouse model were assessed by using microcomputed tomography, histochemical and immunohistochemical, as well as histomorphometry analyses. SiRNA and chemicals were used for gain and loss-of-function assay. Results: In this study, we show that loss of mechanical force led to cellular apoptotic resistance and aging related phenotype, thus reducing the number of apoVs in the circulation due to down-regulated expression of Piezo1 and reduced calcium influx. And systemic infusion of apoVs was able to rescue Piezo1 expression and calcium influx, thereby, rescuing mechanical unloading-induced cellular apoptotic resistance, senescent cell accumulation. Conclusions: This study identified a previously unknown role of mechanical force in maintaining apoptotic homeostasis and eliminating senescent cells. Systemic infusion of mesenchymal stem cell-derived apoVs can effectively rescue apoptotic resistance and eliminate senescent cells in mechanical unloading mice.
Assuntos
Apoptose , Senescência Celular , Vesículas Extracelulares , Animais , Camundongos , Apoptose/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Senescência Celular/efeitos dos fármacos , Senoterapia/farmacologia , Canais Iônicos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Elevação dos Membros Posteriores , Cálcio/metabolismo , Masculino , Estresse MecânicoRESUMO
Cation conducting channelrhodopsins (ChRs) are a popular tool used in optogenetics to control the activity of excitable cells and tissues using light. ChRs with altered ion selectivity are in high demand for use in different cell types and for other specialized applications. However, a detailed mechanism of ion permeation in ChRs is not fully resolved. Here, we use complementary experimental and computational methods to uncover the mechanisms of cation transport and valence selectivity through the channelrhodopsin chimera, C1C2, in the high- and low-conducting open states. Electrophysiology measurements identified a single-residue substitution within the central gate, N297D, that increased Ca2+ permeability vs. Na+ by nearly two-fold at peak current, but less so at stationary current. We then developed molecular models of dimeric wild-type C1C2 and N297D mutant channels in both open states and calculated the PMF profiles for Na+ and Ca2+ permeation through each protein using well-tempered/multiple-walker metadynamics. Results of these studies agree well with experimental measurements and demonstrate that the pore entrance on the extracellular side differs from original predictions and is actually located in a gap between helices I and II. Cation transport occurs via a relay mechanism where cations are passed between flexible carboxylate sidechains lining the full length of the pore by sidechain swinging, like a monkey swinging on vines. In the mutant channel, residue D297 enhances Ca2+ permeability by mediating the handoff between the central and cytosolic binding sites via direct coordination and sidechain swinging. We also found that altered cation binding affinities at both the extracellular entrance and central binding sites underly the distinct transport properties of the low-conducting open state. This work significantly advances our understanding of ion selectivity and permeation in cation channelrhodopsins and provides the insights needed for successful development of new ion-selective optogenetic tools.
Assuntos
Cálcio , Channelrhodopsins , Simulação de Dinâmica Molecular , Sódio , Sódio/metabolismo , Cálcio/metabolismo , Channelrhodopsins/metabolismo , Channelrhodopsins/genética , Channelrhodopsins/química , Animais , Transporte de Íons , Humanos , Células HEK293 , Ativação do Canal IônicoRESUMO
The role of the cerebral cortex in self-initiated versus sensory-driven movements is central to understanding volitional action. Whether the differences in these two movement classes are due to specific cortical areas versus more cortex-wide engagement is debated. Using wide-field Ca2+ imaging, we compared neural dynamics during spontaneous and motorized treadmill locomotion, determining the similarities and differences in cortex-wide activation and functional connectivity (FC). During motorized locomotion, the cortex exhibits greater activation globally prior to and during locomotion starting compared to spontaneous and less during steady-state walking, during stopping, and after termination. Both conditions are characterized by FC increases in anterior secondary motor cortex (M2) nodes and decreases in all other regions. There are also cortex-wide differences; most notably, M2 decreases in FC with all other nodes during motorized stopping and after termination. Therefore, both internally- and externally-generated movements widely engage the cortex, with differences represented in cortex-wide activation and FC patterns.
Assuntos
Cálcio , Locomoção , Córtex Motor , Córtex Motor/fisiologia , Córtex Motor/diagnóstico por imagem , Cálcio/metabolismo , Animais , Locomoção/fisiologia , Masculino , Córtex Cerebral/fisiologia , Córtex Cerebral/diagnóstico por imagem , Feminino , Mapeamento Encefálico/métodos , Camundongos , Caminhada/fisiologiaRESUMO
TRPM4 is a non-selective cation channel activated by intracellular Ca2+ but only permeable to monovalent cations, its activation regulates membrane potential and intracellular calcium. This channel participates in the migration and adhesion of non-excitable cells and forms an integral part of the focal adhesion complex. In neurons, TRPM4 expression starts before birth and its function at this stage is not clear, but it may function in processes such as neurite development. Here we investigate the role of TRPM4 in neuritogenesis. We found that neurons at DIV 0 express TRPM4, the inhibition of TRPM4 using 9-Ph reduces neurite number and slows the progression of neurite development, keeping neurons in stage 1. The genetic suppression of TRPM4 using an shRNA at later stages (DIV2) reduces neurite length. Conversely, at DIV 0, TRPM4 inhibition augments the Cch-induced Ca2 + i increase, altering the calcium homeostasis. Together, these results show that TRPM4 participates in progression of neurite development and suggest a critical role of the calcium modulation during this stage of neuronal development.
Assuntos
Cálcio , Córtex Cerebral , Neuritos , Neurogênese , Canais de Cátion TRPM , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPM/antagonistas & inibidores , Animais , Neuritos/metabolismo , Neuritos/efeitos dos fármacos , Cálcio/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Neurônios/metabolismoRESUMO
Ca2+ release from the sarcoplasmic reticulum (SR) plays a central role in excitation-contraction coupling (ECC) in skeletal muscles. However, the mechanism by which activation of the voltage-sensors/dihydropyridine receptors (DHPRs) in the membrane of the transverse tubular system leads to activation of the Ca2+-release channels/ryanodine receptors (RyRs) in the SR is not fully understood. Recent observations showing that a very small Ca2+ leak through RyR1s in mammalian skeletal muscle can markedly raise the background [Ca2+] in the junctional space (JS) above the Ca2+ level in the bulk of the cytosol indicate that there is a diffusional barrier between the JS and the cytosol at large. Here, I use a mathematical model to explore the hypothesis that a sudden rise in Ca2+ leak through DHPR-coupled RyR1s, caused by reduced inhibition at the RyR1 Ca2+/Mg2+ inhibitory I1-sites when the associated DHPRs are activated, is sufficient to enable synchronized responses that trigger a regenerative rise of Ca2+ release that remains under voltage control. In this way, the characteristic response to Ca2+ of RyR channels is key not only for the Ca2+ release mechanism in cardiac muscle and other tissues, but also for the DHPR-dependent Ca2+ release in skeletal muscle.
Assuntos
Canais de Cálcio Tipo L , Cálcio , Músculo Esquelético , Canal de Liberação de Cálcio do Receptor de Rianodina , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Músculo Esquelético/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Retículo Sarcoplasmático/metabolismo , Sinalização do Cálcio/fisiologia , Acoplamento Excitação-Contração , Modelos Biológicos , HumanosRESUMO
OBJECTIVES: Hyperkalemia is a common life-threatening condition causing severe electrophysiologic derangements and arrhythmias. The beneficial effects of calcium (Ca 2+ ) treatment for hyperkalemia have been attributed to "membrane stabilization," by restoration of resting membrane potential (RMP). However, the underlying mechanisms remain poorly understood. Our objective was to investigate the mechanisms underlying adverse electrophysiologic effects of hyperkalemia and the therapeutic effects of Ca 2+ treatment. DESIGN: Controlled experimental trial. SETTING: Laboratory investigation. SUBJECTS: Canine myocytes and tissue preparations. INTERVENTIONS AND MEASUREMENTS: Optical action potentials and volume averaged electrocardiograms were recorded from the transmural wall of ventricular wedge preparations ( n = 7) at baseline (4 mM potassium), hyperkalemia (8-12 mM), and hyperkalemia + Ca 2+ (3.6 mM). Isolated myocytes were studied during hyperkalemia (8 mM) and after Ca 2+ treatment (6 mM) to determine cellular RMP. MAIN RESULTS: Hyperkalemia markedly slowed conduction velocity (CV, by 67% ± 7%; p < 0.001) and homogeneously shortened action potential duration (APD, by 20% ± 10%; p < 0.002). In all preparations, this resulted in QRS widening and the "sine wave" pattern observed in severe hyperkalemia. Ca 2+ treatment restored CV (increase by 44% ± 18%; p < 0.02), resulting in narrowing of the QRS and normalization of the electrocardiogram, but did not restore APD. RMP was significantly elevated by hyperkalemia; however, it was not restored with Ca 2+ treatment suggesting a mechanism unrelated to "membrane stabilization." In addition, the effect of Ca 2+ was attenuated during L-type Ca 2+ channel blockade, suggesting a mechanism related to Ca 2+ -dependent (rather than normally sodium-dependent) conduction. CONCLUSIONS: These data suggest that Ca 2+ treatment for hyperkalemia restores conduction through Ca 2+ -dependent propagation, rather than restoration of membrane potential or "membrane stabilization." Our findings provide a mechanistic rationale for Ca 2+ treatment when hyperkalemia produces abnormalities of conduction (i.e., QRS prolongation).
Assuntos
Cálcio , Hiperpotassemia , Hiperpotassemia/tratamento farmacológico , Animais , Cães , Cálcio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Eletrocardiografia , Membrana Celular/efeitos dos fármacosRESUMO
As a consequence of hypoosmotic shock, yeast cells swell rapidly and increase the surface area by â¼20% in 20 s. Approximately, 35% of this surface increase is mediated by the ER-plasma membrane contact sites, specifically the tricalbins, which are required for the delivery of both lipids and the GPI-anchored protein Crh2 from the cortical ER to the plasma membrane. Therefore, we propose a new function for the tricalbins: mediating the fusion of the ER to the plasma membrane at contact sites. This proposed fusion is triggered by calcium influx via the stretch-gated channel Cch1 and is supported by the anoctamin Ist2.
Assuntos
Membrana Celular , Retículo Endoplasmático , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Retículo Endoplasmático/metabolismo , Membrana Celular/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Canais de Cálcio/metabolismo , Lipídeos de Membrana/metabolismo , Fusão de Membrana , Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Pressão OsmóticaRESUMO
Sarcomere activation in striated muscle requires both thin filament-based and thick filament-based activation mechanisms. Recent studies have shown that myosin heads on the thick filaments undergo OFF to ON structural transitions in response to calcium (Ca2+) in permeabilized porcine myocardium in the presence of a small molecule inhibitor that eliminated active force. The changes in X-ray diffraction signatures of OFF to ON transitions were interpreted as Ca2+ acting to activate the thick filaments. Alternatively, Ca2+ binding to troponin could initiate a Ca2+-dependent crosstalk from the thin filament to the thick filament via interfilament connections such as the myosin binding protein-C. Here, we exchanged native troponin in permeabilized porcine myocardium for troponin containing the cTnC D65A mutation, which disallows the activation of troponin through Ca2+ binding to determine if Ca2+-dependent thick filament activation persists in the absence of thin filament activation. After the exchange protocol, over 95% of the Ca2+-activated force was eliminated. Equatorial intensity ratio increased significantly in both WT and D65A exchanged myocardium with increasing Ca2+ concentration. The degree of helical ordering of the myosin heads decreased by the same amount in WT and D65A myocardium when Ca2+ concentration increased. These results are consistent with a direct effect of Ca2+ in activating the thick filament rather than an indirect effect due to Ca2+-mediated crosstalk between the thick and thin filaments.
Assuntos
Cálcio , Miocárdio , Animais , Cálcio/metabolismo , Suínos , Miocárdio/metabolismo , Sarcômeros/metabolismo , Miosinas/metabolismo , Troponina/metabolismo , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologiaRESUMO
Vascular calcification is a pathological stage involved in the occurrence and development of cardiovascular diseases, seriously threatening human life and health. At present, few drugs can completely reverse or cure vascular calcification in clinical practice. The pathogenesis of vascular calcification mainly involves the disturbance of calcium and phosphorus homeostasis, autophagy dysfunction, loss of endogenous calcium inhibition, and the apoptosis, cytokine storm, cell osteoblastic transdifferentiation, and stromal vesicle release induced by endoplasmic reticulum stress. Following the therapeutic concepts of warming channels and dredging vessels, activating blood and resolving stasis, tonifying kidney and invigorating spleen, and removing dampness and eliminating turbid, a large number of traditional Chinese medicine(TCM) active compounds/extracts and TCM prescriptions/Chinese patent medicines have shown satisfactory performance in treating vascular calcification, while the specific mechanisms remain unclear and awaits further investigations. This article systematically summarized the pathogenesis of vascular calcification and the latest research progress of TCM in the prevention and treatment of vascular calcification, providing theoretical support for the clinical application of TCM in the prevention and treatment of vascular calcification.